The present invention pertains to image analysis methods used to classify cells based upon their state in the cell grow th and division cycle.
Many interesting bio logical conditions affect or are affected by changes in cell cycle status. For example, some particular condition may cause cells to divide less frequently than normal. Another condition may cause cells to reside in the DNA synthesis state for longer than a normal amount of time. Biological “conditions” of interest to researchers include disease states, normal unperturbed states, quiescent states, states induced by exogenous agents, etc. Valuable insight may be gained by inducing a biological condition through a genetic manipulation, exposure to a particular agent (e.g., a compound, radiation, a field, etc.), deprivation of required substance, and other perturbations.
In drug discovery work, valuable information can be obtained by understanding how a potential therapeutic affects cell growth and division. Often, this information gives some indication of the mechanism of action associated with the compound. For example, a particular class of drugs or genetic manipulations may arrest cell growth at the G2 stage (second gap phase). The drugs of this class are known to act via a particular set of mechanisms of action. Another class of drugs or genetic manipulations arrests cells while in mitosis and acts via a different mechanisms. The ability to quickly determine whether a population of cells is arrested in G2 or mitosis (or some other stage) provides a valuable tool in assessing the mechanism of action of an uncharacterized compound that has been tested on the population of cells.
Commonly, the stage of a given cell in the cell growth and division cycle is determined by measuring the quantity of DNA in the cell. Most cell components are made continuously throughout the so-called “interphase” period, between cell divisions. However, DNA synthesis is an exception. DNA in the cell nucleus is replicated only during a limited portion of the interphase, deemed the “S” phase of the cell cycle (for “synthesis”). The other to distinct stage of the cycle is the cell-division phase, which includes both nuclear division (mitosis) and the cytoplasmic division (cytokinesis) that follows. The entire cell-division phase is denoted as the “M” phase (for “mitosis”). This leaves the period between the M phase and the start of DNA synthesis (the S phase), which is called the “G1” phase (first gap phase), and the period between the completion of DNA synthesis and the next M phase, which is called the “G2” phase. Interphase is thus composed of sequential G1, S, and G2 phases, and can comprise 90% or more of the total cell cycle time.
FIG. 1B is a simple graph depicting how the quantity of nuclear DNA in a cell nucleus changes with cell cycle. As shown, when the cell cycle begins at the G1 phase, the total quantity of nuclear DNA in the cell has a value 2N. That quantity remains constant for the duration of G1. At the onset of the S phase, the total quantity of nuclear DNA begins to increase and steadily grows. By the end of the S phase (beginning of the G2 phase), the total quantity of nuclear DNA has reached a value of 4N. This quantity remains constant throughout the G2 phase and mitosis, until two daughter cells are formed.
Today, cell-cycle analyses are commonly performed using fluorescence-activated cell analysis. This process employs a machine in which a cell suspension is forced through a fine nozzle and an optical measurement is made and recorded for each individual cell as it briefly passes through a window. Initially, a growing cell population is treated with a fixative (to arrest cell division and make the membranes permeable) and contacting them with a dye that becomes fluorescent only when it binds to DNA. When a cell is treated in this way, the intensity with which it fluoresces is approximately proportional to the amount of DNA that it contains. By passing such cells through a fluorescence analyzer, one can rapidly determine the relative fluorescence of the large number of cells, and, therefore, their relative amounts of DNA. Those cells with the least amount of DNA are in the G1 phase, those with double this amount are in the G2 or M phase, while cells in the S phase have intermediate amounts. The lengths of the G1, G2 plus M, and S phases of the cell cycle can be calculated from the fraction of cells in each of these categories. This process is described in detail in various sources including Alberts et al. “Molecular Biology of the Cell” Garland Publishing, Inc. 1993.
Unfortunately, information provided by the fluorescence analyzer is too coarse for many applications. Most importantly, the analyzer only reads out total DNA content per cell; it is unable to distinguish between mitotic cells and G2 phase cells. Therefore, it would be desirable to have an improved process for classifying cells in a manner that distinguishes the interphase and mitotic states.